We have continued our studies on the role of the MyoD family of basic helix-loop-helix muscle-specific gene regulatory factors during myogenic cell commitment and differentiation, analyzing vertebrate muscle formation in vitro or larval muscle formation in developing Drosophila embryos. Vertebrates express four different myogenic factors in a developmentally regulated pattern whereas Drosophila has only a single related factor. In vertebrates, MyoD and myogenin are the two factors expressed in cultured muscle. We have shown that the putative auto regulation of the avian MyoD(CMD1) promoter is indirect and does not involve the direct binding of the bHLH myogenic factors or MEF2. The myogenic bHLH proteins are nuclear phosphoproteins. We have identified a regulatory serine residue in the carboxy terminal portion of CMD1 that is a potential casein kinase II site and plays a role in the homo-heterodimer equilibrium of CMD1 and the ubiquitous E- protein, E12. The same site is conserved in Drosophila MyoD. The myogenic factors bind DNA predominately as heterodimers with the E2A- related proteins, E12 and E47 but little is known about the in vivo complex. We have shown that MyoD and myogenin are complexed in muscle cultures with E2A proteins and that myogenesis can be regulated depending upon the level of the E2A gene products using combinations of sense and antisense E12 RNA expression vectors. We have previously shown, by comparison with Drosophila MyoD, that dimerization specificity within the vertebrate myogenic factors and E proteins is determined, to a large extent, by the nonconserved nonhydrophobic residues in the HLH domain. We have extended this study to further demonstrate conserved glutamate residues in helix 2 of MyoD are required to overcome the inhibitory domain of E12 and that this inhibitory domain blocks E12 homodimerization and not DNA binding, as previously proposed. The role of dimerization specificity in the regulation of the myogenic factors has been demonstrated directly in vivo and in vitro: we have been able to rescue the lethal C. elegans MyoD point mutation, HLH1, with Drosophila MyoD and, to a lesser extent, with avian myogenin; conversion of mouse 10T1/2 fibroblasts to muscle only works with Drosophila MyoD when it is paired with daughterless, the Drosophila homolog to the E-proteins, or the C. elegans E-protein homolog. The Drosophila MyoD alone will not activate myogenesis in mouse cells which demonstrates the homodimer is not functional and is consistent with the results of the E12 antisense experiments.